The present invention relates to antibodies against molecules, particularly haptens and other B cell antigens, and methods for their preparation. In the methods of the present invention, the molecule is conjugated to a carrier comprising a mixture of charge-modified proteins prior to raising of the antibodies in a host animal. The host animal is pre-immunized with unmodified proteins of the carrier. The host animal is then immunized with the molecule complexed with the carrier wherein proteins in the mixture making up the carrier have been charge-modified. Antibodies or B cells producing antibodies can then be routinely isolated from the host animal. The present invention also relates to methods for use of these antibodies in immunoassays as well as in therapeutic applications. In addition, the present invention relates to vaccine therapies and methods of invoking and/or enhancing an immune response in animals via molecules coupled to carriers comprising a mixture of charge-modified proteins.
The general humoral immune response is based on the cooperation of antigen presenting cells, antigen specific T lymphocytes and antigen specific B lymphocytes.
T lymphocytes are selected and stimulated by T cell epitopes, which associate with Major Histocompatibility Complex (MHC) and are presented on the surface of antigen presenting cells. A T cell epitope is a short, linear amino acid sequence most frequently localized to the interior portion of a protein and thus inaccessible from the protein surface. A T cell epitope reacts with the antigen specific receptor of the T cell only when bound to an MHC molecule thus forming part of a ternary complex. Helper T cells participate in determining what kinds of immunoglobulins are produced by B cells and stimulate the proliferation of specified lymphocytes. Peptides arising from the digestion of internalized and endocytosed proteins by a B cell are bound to MHC proteins and stimulate and activate helper T cells.
Accordingly, these T cell epitopes react with the antigen specific receptor of the helper T cell only when the helper T cell epitope is bound to an MHC Class II molecule.
B lymphocytes are selected by antigens which bind to the antigen specific receptor (a membrane bound antibody) of the B cell. Almost any molecular configuration can act as a B cell antigen or B cell epitope. With respect to proteins, the epitopes are most frequently positioned on the outer accessible face of the protein as direct binding to the antigen specific receptor is required. The T cell epitope of the antigen is then presented on the surface of the B cell, associated with MHC class II molecules. MHC class II proteins, which comprise the T cell epitopes, arise from the degradation of proteins that have been internalized. This presentation entails the selected and stimulated T accessory cells to be associated with the antigen specific B lymphocyte, which is now stimulated and matured via hormone like factors from the T cell. The final stage of the B cell after stimulation and maturing is the antibody producing plasma cell, which secretes antigen specific antibody (Biochemistry ed. Lubert Stryer 4th Edition, Freeman and Co., New York pp. 379).
Thus, antibody formation against an antigen is conditional upon the presence of both B cell epitopes and T cell epitopes. Further, it has been demonstrated that the two types of epitopes must be physically associated to generate an antibody immune response (Rajewsky et al. J. Exp. Med. 1969 129:1131; Mitchison, N. A. Eur. J. Immunol. 1971 1:10; and Mitchison, N. A. Eur. J. Immunol. 1971 1:18).
Accordingly, the antibody response to low molecular weight molecules such as haptens is often quite poor and can be induced in a mammal only by presentation of the low molecular weight molecule coupled to an immunogenic carrier. Coupling to an immunogenic carrier is also required for relatively large molecular weight molecules with only limited immunogenicity such as larger peptides, low immunogenic proteins (such as self-proteins), carbohydrates, lipids, viruses and nucleic acids. Coupling to an immunogenic carrier also provides a means for enhancing the immunogenicity of immunogenic proteins wherein a more powerful immunogenicity is required. Some examples of carrier molecules which have been disclosed for inducing or enhancing an immune response to such molecules include serum albumin such as bovine serum albumin (BSA), human serum albumin (HSA) and others, keyhole limpet hemocyanin (KLH), ovalbumin (OA), chicken immunoglobulin (IgY) and diphtheria toxoid.
Conjugation methods have also been described for increasing the immunogenicity of low molecular weight molecules. Pioneering work to enhance the immunochemistry of low molecular weight molecules began as early as 1917 when antigens were covalently conjugated by azo reaction on histidine, tyrosine and tryptophan residues (Landsteiner, K. The Specificity of Serological Reactionsxe2x80x9d Harvard Univ. Press, Cambridge, Mass., 1945). More recently, methods have been described for preparing hapten-carrier conjugates via formation of covalent bonds between the low molecular weight hapten and a carrier protein selected from a wide range of globulin fractions (Erlanger, B. F. The Preparation of Antigenic Hapten-Carrier Conjugates: A Surveyxe2x80x9d in Methods of Enzymology, 70, Academic Press, Inc. 1980). In these methods, the functional groups of the hapten determine which synthesis is selected for conjugation of the functional groups of the carrier to the antigen. A wide range of chemical syntheses have been described for conjugating hapten on the basis of hapten functionality.
In addition to invoking an antibody response, it is also desirable to avoid antigen-specific suppression wherein prior immunity against a carrier protein reduces the response to re-inoculation with the same carrier protein coupled to an antigen. This effect can be avoided or reduced, enabling expansion of native B cell populations, if the carrier molecules, for example secretory proteins from BCG, are sufficiently modified at the surface. Reduction of B cell response to the carrier molecule can be achieved by chemical or physical modification of the carrier molecule. Examples of chemical and/or physical processes, which have been described for modification of the carrier molecule include heat denaturation and chemical methylation or formaldehyde treatment of the carrier molecule prior to the coupling to the hapten molecule.
Modification of the carrier to avoid epitope-specific suppression has several additional advantages. Specifically, since T cell immunity can be induced as a general principal in animals, the T cell immunity can be retrained against the modified carrier while considerably reducing the degree of B cell immunity against the carrier molecule.
Accordingly, subsequent immunization with a B cell antigen coupled to a modified form of the carrier ensures a rapid development of antibodies, a high level of antibodies after only one or two immunizations, a sustained immune response, and the possibility of rapid development of antibodies having a high binding strength.
WO 94/12213 describes a process for modifying an immunogenic protein to be exclusively or predominantly a T cell antigen by blocking charged groups, either xe2x80x94NH3+ or xe2x80x94COOxe2x88x92. xe2x80x94NH3+ groups are blocked by means of formaldehyde, while disulfide bridges are reduced by means of dithiothreitol or xcex2-mercaptoethanol. Denaturation of the protein antigen is then performed by heat treatment.
The present invention relates to a new method for raising antibodies against molecules with low antigenicity by conjugating the molecule to a carrier comprising a balanced charge mixture of proteins chemically modified to carry a net positive or negative charge.
An object of the present invention is to provide a method for raising antibodies against molecules including, but not limited to, those with low immunogenicity. The method comprises coupling the molecule to a carrier comprising a balanced charge mixture of proteins, each protein of the mixture being chemically modified to carry either a net positive or negative charge. In this method a host animal is pre-immunized with unmodified proteins of the carrier or the host attenuated bacteria or virus from which the carrier proteins are derived. The host animal is then immunized with the molecule coupled to a carrier comprising a balanced charge mixture of the proteins, each protein of the mixture being chemically modified to carry either a net positive or negative charge. Antibodies raised against the molecule by the host animal can then be isolated in accordance with well known methods.
Another object of the present invention is to provide antibodies raised in accordance with this method.
Another object of the present invention is to provide immunoassays and immunoassay kits, which use antibodies raised in accordance with this method.
Another object is to provide pharmaceutical compositions comprising antibodies raised in accordance with this method, as well as methods of using these pharmaceutical compositions in therapeutic applications.
Yet another object of the present invention is to provide vaccines and methods of inducing an immune response in animals by administering vaccines comprising either an antibody raised in accordance with the method of the present invention or a molecule coupled to a carrier comprising a balanced charge mixture of proteins, each protein of the mixture being chemically modified to carry either a net positive or negative charge.
The present invention relates to a complex carrier system for generating antibodies. In this system, the carrier molecule is modified by chemical treatment, preferably via formaldehyde treatment and acylation of a protein mixture, to make the product carry a net positive or negative charge. Accordingly, in this technique, the protein mixture is denatured by treatment that removes a particular group that is necessary to form a hydrogen bond with another group on the protein. A second batch of protein is also prepared with the other group modified. Through subsequent combination of the two different groups, equalization of the opposite charges is achieved and a charge balance is obtained. This process results in the production of an electrically neutral carrier molecule complex with pertinent advantages for fulfillment of the objective of raising specific antibodies in an efficient manner. Specifically, the carrier molecule complex retains the identity characteristics of the original component proteins while constituting a conjugate that has increased greatly in size and complexity.
For purposes of the present invention, the carrier molecule to be modified is an immunogenic protein. For example, the carrier molecule may comprise protein molecules such as serum albumin, e.g., BSA (bovine serum albumin) and HSA (serum albumin), OA (ovalbumin) or mixtures thereof, e.g. BSA (positive charge) and OA (negative charge). Suitable immunogenic proteins for carrier molecules can also be derived from intracellular bacteria including, but not limited to, Salmonella sp., Ricketsia, Chlamydia, Brucella, Mycobacter tuberculosis, Mycobacter leprae, Listeria monocytogenes, Leishmania donovni, Plasmodium vivax and Franasella tularensis. Suitable materials for carrier molecules can also be derived from organisms such as viruses. Examples of various immunogenic proteins for use in the present invention are also described in U.S. Pat. No. 5,955,077, WO 94/12213 and WO 89/06974.
A preferred protein mixture useful in the present invention is the SP (secretory protein) from intracellular bacteria. In this embodiment, a crude filtrate from intracellular bacteria is divided into two portions, the first portion being treated with formaldehyde to produce a portion carrying a net negative charge, the second portion being esterified by acylation to produce a portion carrying a net positive charge. In a preferred embodiment of the present invention, the SP carrier compound used for the preparation of antibodies is derived from Bacille Calmette-Guerin (BCG) However, as will be obvious to those of skill in the art upon this disclosure other carrier compounds can also be used.
There are several advantages in using a carrier complex of mixed SP products from Bacille Calmette-Guerin (BCG) after treatment by esterification or with formaldehyde to raise antibodies against a molecule with low immunogenicity. Specifically, particulate, or sparingly soluble complexes are formed between the positively charged esterified, methylated protein solution and the negatively charged formaldehyde treated solution. These complexes are more immunogenic than the individual soluble carrier protein materials. In addition, there is an adjuvant effect related to the non-covalent binding nature of the bonding of the complexes formed by combining the positively and negatively charged proteins. This bonding results in slow release of the single molecule-carrier conjugates which is similar to that obtained with Al(OH)3 adjuvant. The ability to adjust and control the charge balance by changing the relative proportions of the positive and negative charged SP portions also enables optimization of the immunogenic effect. Further, because of the increased size of the complexes from non-specific coupling, the ability of antigen presenting cells to take up the complexes and transport them to the local lymph node is enhanced.
As will be obvious to those of skill in the art upon this disclosure, other methods for obtaining positive charged and negative charged proteins fractions can also be used. Other methods of obtaining positive and negative charged proteins are described, for example, by Erlanger, B. F. (The Preparation of Antigenic Hapten-Carrier Conjugates: A Surveyxe2x80x9d in Methods of Enzymology, 70, Academic Press, Inc. 1980) and WO 94/12213.
The coupling of a molecule with low antigenicity to a carrier to form a complex comprising the coupled molecule and carrier can be performed either covalently or non-covalently. Coupling of a hapten or B cell antigen can be achieved by mixing the positively charged protein mixture with the negatively charged protein mixture and with the hapten. Simple mixing of these compounds results in formation of non-covalent bonds between the molecules.
Alternatively, the hapten can be covalently coupled to either the positive or negative protein mixtures before combining the protein mixtures. Various methods for covalent coupling are known and selection of the method is based upon the molecule being coupled to the carrier. For example, coupling of a molecule with sulfhydryl groups can be performed by homobifunctional coupling with the 1,4-bis-maleimidobutane (BMB) reagent. Coupling of a carbohydrate molecule can be performed using sodium periodate. Coupling of a molecule with carboxyls can be performed using 1-ethyl-3-(3-dimethylaminopropyl) carboiimide hydrochloride. Coupling of a molecule with hydroxyls to sulfhydryls can be performed using N-(p-maleimidophenyl) isocyanate. Alternatively, non-selective/photoreactive coupling can be performed using N-5-azido-2-nitrobenzoyloxysuccinimide. Covalent coupling of a molecule to the carrier can also be achieved via glutaraldehyde coupling.
Glutaraldehyde is a dialdehyde homobifunctional cross-linker that provides covalent bonds to amino-groups on both hapten and carrier proteins. The reaction between one end of the dialdehyde glutaraldehyde and a primary amine is termed as nucleophilic addition-elimination resulting in the formation of an imine group thus binding the glutaraldehyde. Specific methods for glutaraldehyde coupling a hapten to a carrier protein of the present invention are set forth in Example 5.
In yet another embodiment the molecule is coupled to the carrier to form the complex comprising the molecule coupled to the carrier after the two protein mixtures are combined.
One advantage in conjugating carriers of both positive and negative charged proteins to molecules with low antigenicity is the effect upon immunization as the large complexes are engulfed by macrophages and other antigen presenting cells. Further, T cell epitopes are not destroyed by the chemical modifications. In addition, the modified carriers of the present invention have an activating effect on the immune response as the reformed hydrogen bridges between xe2x80x94NH2 nuclei and COOH allow for formation of large complexes which are engulfed and transported to the lymph nodes more readily by macrophages and other antigen-presenting cells. Macrophages and other antigen presenting cells which engulf the complex recognize features of the complex that are unknown or not previously presented, i.e. the complexed hapten. However, the similarities of the antigenic features of the carrier with the pre-immunization antigen are also recognized. T cell epitopes on the complex are not destroyed or modified by chemical treatment and their high levels results in somatic hypermutation thereby resulting in antibodies with much higher affinity.
Once formed, the complex is administered to a host animal so that antibodies are produced against the molecule and/or B cells producing antibodies against the molecule are produced. Antibodies can be isolated from these host animals in accordance with well known techniques. B cells can also be isolated and used to produce hybridomas in accordance with well known techniques.
Pre-immunization of the host animal with proteins used in the complex of the present invention is required. However, in this pre-immunization step the charge of proteins is not modified. These proteins are referred to herein as xe2x80x9cunmodified proteinsxe2x80x9d. By xe2x80x9cunmodified proteinsxe2x80x9d it is meant to include not only the proteins used in the carrier but also the entire intracellular bacteria or virus or attenuated bacteria or viruses from which the charge modified proteins of the carrier are derived. In a preferred embodiment, the unmodified proteins used in the pre-immunization step comprise the entire intracellular bacteria or virus or attenuated bacteria or virus. The advantages of pre-immunization with the protein or proteins followed by immunization with the complex containing modified proteins of the present invention are that pre-immunization generates a high incidence of specific T cells. See e.g. U.S. Pat. No. 5,955,055. Because the immune response is a T cell response there will be a high degree of somatic hypermutation in comparison with methods with no pre-immunization step. Examples of host animals used to produce antibodies include, but are not limited to, rodents, lagomorphs, equines, bovines, ovines, canines, felines, porcines and the like.
In a preferred embodiment, the antibodies of the present invention are human or humanized. Various methods for producing humanized antibodies have been described. In one embodiment, antibodies can produced against the carrier coupled molecule in animals genetically modified to produce humanized antibodies. Transgenic mice for production of humanized antibodies are known in the art. Antibodies can be isolated from these host animals in accordance with well known techniques.
In another embodiment, humanization is performed via reshaping or complementarity determining region (CDR) grafting. These techniques for humanization are well-established and reduce the immunogenicity of monoclonal antibodies from other animal species in humans. These techniques can be used to humanize antibodies produced from existing animal antibody-producing cell lines such as mouse antibody-producing cell lines. In CDR grafting, the antigen-binding region of a mouse antibody, for example, is genetically engineered onto the immunoglobulin framework of a human antibody. More specifically, genes encoding the antigen binding regions (CDRs) of the mouse antibody are isolated and merged with the genes encoding a normal human antibody. The result is a human antibody with mouse antigen binding regions. This technique offers a means for introducing a novel antibody into a human while bypassing the normal anti-idiotypic immune response to introduction of a foreign protein.
Humanization of antibodies of the present invention can also be obtained via phage display or from human monoclonal clones in accordance with well known techniques.
The carriers of the present invention are particularly useful in inducing an antibody response against molecules with low antigenicity. Examples of molecules with low antigenicity include, but are not limited to, low molecular weight haptens and higher molecular weight molecules with low antigenicity such as larger peptides, low immunogenic proteins, carbohydrates, lipids and nucleic acids. Examples of haptens or low molecular weight molecules against which antibodies can be raised in accordance with the method of the present invention include, but are in no way limited to, 2,6 dichlorobenzamide, triazines and other pesticides, antimalarial drugs as well as other low molecular weight drugs, and steroid hormones. Examples of proteins with low immunogenicity against which antibodies can be raised in accordance with the method of the present invention include, but are in no way limited to, interleukins, immunoglobulins, cell markers, albumins, peptide hormones, and prions. Examples of carbohydrates against which antibodies can be raised in accordance with the method of the present invention include, but are in no way limited to, glycogen, cellulose, glucose, maltose, lactose and cellobiose. Examples of lipids against which antibodies can be raised in accordance with the method of the present invention include, but are in no way limited to, lipopolysaccharides, triglycerols, lipid membranes, phospholipids, liposomes, and fatty acids.
However, as will be understood by those of skill in the art upon reading this disclosure, the method of the present invention can also be used to enhance immunogenicity of highly immunogenic proteins. Examples of highly immunogenic proteins against which antibodies can be raised in accordance with the method of the present invention include, but are in no way limited to, bacterial and viral proteins, surface proteins of non-self cancer cells, plant proteins, gluten, milk proteins, and bacterial exotoxins. Antibodies can be raised against proteins of bacteria including, but certainly not limited to, Streptococci, Staphylococcus aureus, E. coli, Pneumococci, Salmonella, and Borellia, and viruses including, but certainly not limited to, cytomegalovirus, Epstein Barr Virus, HIV, Influenza, and Hepatitis.
Using the method and carrier system of the present invention, antibodies were raised against the hapten, chloroquine. Chloroquine is a common anti-malarial drug sold over the counter in many countries. Due to its widespread availability, an increase in both unintentional intoxications and attempted suicides with this compound has occurred. The toxic effects of chloroquine are blurred vision, vertigo, tinnitus, nausea, shock, convulsions, coma, heart arrhythmias, and respiratory inhibition. In cases of overdose, gastric lavage is performed as well as treatment with the vasoconstrictor diazepam and sometimes assisted ventilation. Ineffective compliance and use of subtherapeutic doses of chloroquine actually increases the risk for contracting malaria. In fact, inefficient use of chloroquine in malarious areas has been suggested to provoke the appearance of chloroquine resistant P. faliparum. Thus, the ability to monitor chloroquine levels in non-infected and infected patients would be useful, especially in children and patients with malabsorption and gastrointestinal disorders. Several calorimetric field methods for measuring chloroquine levels are available. The Dill-Glazko test was used widely until recent reports of unreliability. Other field methods include the Saker-Solomon, the Haskins and the Bromothymol Blue method. While these tests perform better than the Dill-Glazko test, they still lack sensitivity and specificity. The most reliable method for chloroquine detection is via HPLC. However, HPLC methods are time consuming, labor intensive and can only be performed in well-equipped laboratories. Accordingly, there is a need for a low cost, reliable and quick field method of chloroquine measurement which is satisfied with antibodies raised against chloroquine using the systems and methods of the present invention.
Using the method of the present invention, antibodies against chloroquine were raised against the side chain to the quinoline moiety of chloroquine, 2-amino-5-diethylaminopentane (ADP). ADP contains a primary amine which is reactive in glutaraldehyde coupling reactions. Further, this is the portion of the chloroquine molecules which antibodies preferably recognize in order to avoid cross reactivity towards metabolites of chloroquine and other malarial drugs.
A detailed protocol of experiments in mice administered the ADP antigen is provided in Example 7. The experiments demonstrated that mice (n=5) administered ADP in combination with SP +/xe2x88x92 in accordance with the method of the present invention exhibited higher antibody production as early as the second bleed as compared to mice administered ADP and SP which was not charge modified. Further, comparison of the antibody titer between the first and second bleed in each group showed mice treated with ADP in combination with +/xe2x88x92 SP to exhibit an 8.4-fold increase in antibody titer while mice receiving ADP in combination with SP which was not charge modified exhibited only a 4.9-fold increase in antibody titer.
Antibodies raised against a molecule in accordance with the method of the present invention can be incorporated into immunoassay kits for detection of the molecule. The immunoassay kits of the present invention comprise these antibodies. In a preferred embodiment, the antibodies are detectably labeled. Examples of detectable labels include, but are not limited to, enzymes, fluorophores and radiolabels. Immunoassay kits of the present invention may further comprise detection means for the labeled antibodies, standards, and dilution and/or washing buffers.
Antibodies raised in accordance with the method of the present invention and isolated from a host animal can also be used in vivo as diagnostic imaging agents and incorporated into pharmaceutical compositions for protection against and treatment of various infections and diseases. The use of antibodies for in vivo diagnosis and treatment is well known in the art. For example, antibody-chelators labeled with Indium-111 have been described for use in the radioimmunoscintographic imaging of carcinoembryonic antigen expressing tumors (Sumerdon et al. Nucl. Med. Biol. 1990 17:247-254). In particular, these antibody-chelators have been used in detecting tumors in patients suspected of having recurrent colorectal cancer (Griffin et al. J. Clin. Onc. 1991 9:631-640). Antibodies with paramagnetic ions as labels for use in magnetic resonance imaging have also been described (Lauffer, R. B. Magnetic Resonance in Medicine 1991 22:339-342). Antibodies raised in accordance with the method of the present invention can be used in a similar manner.
In this embodiment, it is preferred that the host animal from which the antibodies are derived be transgenically modified to produce antibodies compatible with the target animal. Alternatively, following production the antibody can subsequently be modified for compatibility with the target animal.
Pharmaceutical compositions comprising an antibody or a part thereof produced in accordance with the method of the present invention can be formulated in accordance with well known techniques. Parts of antibodies preferred for use in the present invention include, but are not limited to Fab fragments and Fv fragments. Pharmaceutically acceptable vehicles for formulation of compositions comprising an antibody are well known and can be selected routinely by one of skill in the art based upon the selected route of administration for the pharmaceutical composition. For example, for intravenous administration, pharmaceutical compositions of the present invention will preferably comprise an antibody produced in accordance with the method of the present invention in a pharmaceutically acceptable vehicle such as phosphate buffered saline.
Pharmaceutical compositions comprising an antibody or part of an antibody produced in accordance with the method of the present invention can be administered to a subject in need thereof in accordance with well known procedures to treat various diseases and/or infections.
In addition, carriers of the present invention coupled to a selected molecule can be used as vaccines to invoke an immune response against the selected molecule. These vaccines comprise an immunogenically stimulatory amount of the carrier coupled to the selected molecule. Immunogenically stimulatory amount refers to that amount of carrier coupled molecule that is able to invoke the desired immune response in the recipient for the protection against, amelioration, or treatment of the disease for which the vaccine is being administered. Effective amounts may be determined empirically by standard procedures well known to those skilled in the art. In this embodiment, the vaccine therapy further comprises a pre-immunization step wherein the recipient is administered unmodified proteins of the carrier prior to immunization with the molecule coupled to the carrier of proteins which have been chemically modified to carry either a net positive or negative charge.
The antibody or carrier coupled molecule may be provided in any one of a number of vaccine formulations which are designed to induce the immune response. Such formulations are known in the art and include, but are not limited to, formulations such as those described in U.S. Pat. No. 5,585,103. Vaccine formulations of the present invention used to stimulate immune responses can also include pharmaceutically acceptable adjuvants such as Al(OH)3.
The following nonlimiting examples are provided to further illustrate the present invention.